The disclosure relates, in general, to the iterative detection of biological molecules and, more particularly, to cleavable linkers for tethering detectable moieties to biological probes.
Fluorescence imaging is a pervasive technique in the fields of chemistry, biology, medicine and so forth. For example, comprehensive protein profiling on the single-cell level may be useful to understand complex molecular pathways in heterogeneous cell populations (Bendall, et al., Nat. Biotechnol. 2012, 30, 639; Bendall, et al., Science 2011, 332, 687; Bodenmiller, et al., Nat. Biotechnol. 2012, 30, 858; Ma, et al., Nat. Med. 2011, 17, 738; Wei, et al., Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E1352; Shi, et al., Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 419). Immunofluorescence microscopy has been widely used to quantify the identities, positions and abundances of proteins in individual cells. Therefore, it may be well-suited for analyzing cell heterogeneity and the spatial complexity of proteins. Similarly, fluorescence based detection schemes have been developed for DNA and RNA-based targets. However, due to the spectral overlap of commonly available fluorophores, the capacity of conventional immunofluorescence techniques can be limited (Guo, J. et al., Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 3493).
Various techniques have been developed to address limitations of conventional fluorescence imaging techniques and enable more comprehensive DNA, RNA and protein analysis. One class of techniques includes multiplex or iterative immunofluorescence with continuous cycles of staining, imaging, and fluorescence signal erasing. Example methods used for iterative immunofluorescence may rely on photobleaching (Schubert, et al., Nat. Biotechnol. 2006, 24, 1270), chemical reagents (Gerdes, et al., Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 11982; Micheva, et al., Neuron 2007, 55, 25; Micheva, et al., Neuron 2010, 68, 639; Zrazhevskiy, et al., Nat. Commun. 2013, 4, 1619) or DNA displacement reactions (Schweller, et al., Angew. Chem. Int. Ed. Engl. 2012, 51, 9292; Duose, et al., Nucleic Acids Res. 2012, 40, 3289; Duose, et al., Bioconjug. Chem. 2010, 21, 2327) to photobleach fluorophores, chemically inactivate fluorophores, remove fluorescent antibodies or disassemble fluorescent DNA complexes.
While each of the aforementioned methods may offer advantages over conventional fluorescence imaging techniques, a number of limitations remain. In one aspect, partial photobleaching during the imaging process and incomplete fluorescence signal removal after bleaching may hinder the consistent quantification of protein abundances. In another aspect, harsh chemical reagents often result in specimen degradation and interfere with subsequent cycles of staining. In a further aspect, mis-hybridization between different DNA complexes along with non-specific binding between DNA complexes and endogenous biomolecules may lead to increased background. Recently, mass cytometry imaging (Giesen, et al., Nat. Methods 2014, 11, 417) and ion beam imaging (Angelo, et al., Nat. Med. 2014, 20, 436) with metal isotope labeled antibodies have been developed for protein profiling. However, these mass spectrometry based approaches may have limited imaging resolution or imaging speed and sample throughput. Accordingly, there is a need for fluorescence imaging techniques that overcome the aforementioned limitations.